ANTIBIOTIC RESISTANCE SURVEILLANCE OF HELICOBACTER PYLORI AT THE BIOBÍO REGION (CHILE) IN A DECADE

ABSTRACT BACKGROUND: Helicobacter pylori infection in Chile remains as a public and private health-care system’s challenge, with a prevalence of the infection over 70%. Nowadays, antibiotic treatment of the infection is mandatory to prevent the arising of severe associated diseases but failures in the eradication therapy mainly due to clarithromycin resistance has been observed worldwide and first line eradication therapy seems to be not effective anymore in several geographical areas. Thus, health-care systems are committed to maintain an epidemiological surveillance upon the evolution of the antibiotic resistance of this priority 2 pathogen. OBJECTIVE: This work reports a 10 years surveillance of the primary antibiotic resistance of H. pylori clinical isolates at the Biobío region-Chile, and the evolution of resistance toward amoxicillin, clarithromycin, levofloxacin, metronidazole, and tetracycline among the species. METHODS: H. pylori strains were investigated during the periods 2005-2007 (1435 patients analysed) and 2015-2017 (220 patients analysed) by inoculating a saline homogenate biopsy onto the surface of Columbia agar (Oxoid, Basingstoke, UK) - supplemented with 7% horse red blood cells plus DENT inhibitor (Oxoid, Basingstoke, UK) - following by incubation at 37ºC under 10% CO2 atmosphere for five days. Antibiotic resistance pattern of the isolates was assessed using the disk diffusion test in Müeller-Hinton agar supplemented with 7% horse red blood cells followed by incubation for further three days under 10% CO2 atmosphere. Statistical analysis was done using the SPSS v22 software and P values <0.05 were considered statistically significant. RESULTS: A total of 41% of 1435 patients were detected to be infected with H. pylori by bacteriological culture in 2005-2007 period, meanwhile 32.7% from 220 patients were also infected in 2015-2017 period. The clinical isolates of H. pylori are mostly susceptible to amoxicillin and tetracycline (both over 98% of strains), but less susceptible to levofloxacin in both periods analysed (over 79% of the strains). On the other hand, metronidazole continuous showing the highest score of resistant isolates (over 40% of resistant strains), although an 18% fewer resistant strains were observed in 2015-2017 period. Clarithromycin, the key antibiotic in eradication therapies, has an increased frequency of resistant strain isolated in the decade (22.5% in 2005-2007 and 29.2% in 2015-2017). Multidrug resistant strains (two, three and four antibiotics) were also detected in both periods with the highest scores for simultaneous resistance to clarithromycin-metronidazole (18%) and clarithromycin-metronidazole-levofloxacin (12.5%) resistant strains. According to gender, the isolates resistant to amoxicillin, clarithromycin and metronidazole were more frequent in female, with a specific increment in amoxicillin and clarithromycin resistance. CONCLUSION: The frequency of clarithromycin resistance (29.2%) detected in 2015-2017 suggests that conventional triple therapy is no longer effective in this region.

In vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody.

BALB/c mice were injected intravenously with three different monoclonal antibodies (mAbs) specific for complement receptor 1 (CR1). Two of the mAbs crossreacted with CR2. 24 h later, the mice were immunized with horse erythrocytes or keyhole limpet hemocyanin (KLH), and the primary antibody response was measured. One of the anti-CR antibodies, 7G6, suppressed greater than 99% of the direct plaque-forming cell response against horse red blood cells (HRBC). The same antibody markedly suppressed the serum antibody responses to both HRBC and KLH. To be optimally suppressive, the mAb had to be injected before suboptimal concentrations of antigen. The other two complement receptor-specific antibodies had very moderate, if any, effects on the antibody response. 7G6 was able to downregulate CR1 and CR2 on the surface of B cells and, in addition, to inhibit rosette formation with C3d-coated sheep erythrocytes (EC3d). One of the antibodies with a weak effect downregulated only CR1. The other downregulated both CR1 and CR2, although not as efficiently as 7G6, and was unable to inhibit EC3d rosette formation. We conclude that the reason 7G6 is outstanding in its suppressive capacity is that it is the only mAb tested that functionally blocks CR2. The data suggest that CR2 is of crucial importance in the initiation of a normal antibody response to physiological concentrations of antigen.

Purification and use of limulin: a sialic acid-specific lectin.

A simple and rapid method for the isolation of the sialic acid-specific lectin, Limulus polyphemus hemagglutinin (LPA), from the hemolymph of Limulus polyphemus is described. Declotted hemolymph is adsorbed to an affinity chromatographic column consisting of hog gastric mucin glycopeptides coupled to agarose and LPA is eluted in a single step with a Ca2+-free buffer, giving an apparent purification of approximately 25,000-fold. The eluted material is homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing and consists of identical subunits each of 29,000 daltons. Hemagglutination inhibition studies with horse red blood cells indicate specificity of LPA for N-acetyl- and N-glycolylneuraminic acid; binding is Ca2+ -dependent and abolished by neuraminidase treatment. LPA was covalently coupled to rhodamine and to horseradish peroxidase for use in detection of sialoglycoconjugates on cells and tissues by light and electron microscopy. Examples of the use of LPA for detection of sialoglycoconjugates in rat renal tubules and glomeruli, blood vessels in rat pancreas, and on horse red blood cells are shown. The procedures described here should prove useful as a cytochemical probe for detection of sialoglycoconjugates in a variety of systems. An accompanying article utilizes these probes for the detection of sialoglycoconjugates on the plasmalemma of adult and differentiating rat pancreatic acinar cells.